Non-Chromatographic Methods for Macromolecule Isolation and Purification
Producers of plasma proteins from natural sources have long depended on ethanol based differential protein precipitation to sequentially isolate protein fractions prior to purification of individual proteins via column chromatography. Such approaches are particularly suited to larger scale separations run with tight production economies. However, they are technically complex and require highly trained technicians and expensive equipment and facilities.
Scientists involved in biotechnical separation processes are dealing with ever larger processes and the need to reduce the costs of produced goods. They are re-examining the use of techniques such as precipitation and partitioning in aqueous two-phase systems (1, 2, 3). The main advantage of partition is that it is based on formation of two liquid (L) phases such that cells and cell debris can be collected at the L-L phase interface where they are held by interfacial tension. Thus if target protein can be partitioned to a significant degree into one of the phases, in a selective manner, it is possible to effect some degree of target clarification (cell debris removal), purification, and concentration all in one step (above refs.). The major disadvantage to partition is that formation of aqueous polymer two phase systems typically require adding either two polymers such as dextran and polyethylene glycol [PEG] or adding one polymer and a high concentration of salt (e.g. PEG and 0.5M phosphate) to target containing solutions. The two polymer systems can be expensive and the polymer-salt systems have low capacity (solubility)—often 1 to 2 g/L for antibody proteins (3). An approach to clarify biotechnical feeds by adding relatively low concentrations of a single biocompatible thermotropic polymer has recently been disclosed (4), which allows for clarification via formation of an essentially polymer-free phase, which contains most protein, and typically floats on top of a denser polymer-rich lower phase. The upper phase can also be modified via addition of precipitating agents so as to effect further concentration and purification of target.
Single Polymer Aqueous Two-Phase Systems
It has long been known that proteins can be selectively partitioned between the phases of aqueous polymer two phase systems formed spontaneously using two polymers such as dextran and polyethylene glycol or one polymer such as PEG and relatively high concentrations of a water structuring salt such as NaPhosphate. The latter systems are less expensive however they tend to have lower protein solubility and thus capacity (3, 4). It has also long been appreciated that cells and cell debris, and other micron sized colloids, tend to accumulate at the interface of the two phases where they are held by interfacial tension. One variant of the above two types of LL systems has been polymer-polymer systems where one polymer is a thermoresponsive polymer which self associates under elevated temperature and other conditions (3, 4). This behaviour has previously been used, following phase separation and isolation of the two phases, to isolate proteins from polymer in one of the phases, and recover phase polymer for re-use. In addition to those two types of phase systems a third type has been recognized—single polymer systems. These are formed under conditions where added hydrophilic polymer self-associates and in so doing forms a lighter polymer-poor phase floating on top of a much denser polymer-rich phase (4, 5). Suitable polymers include so called “EOPO” polymers (e.g. Pluronic®, Tergitol®, Breox® families) made from ethylene oxide (EO) and propylene oxide (PO) components. Other suitable polymers may include cellulose or other polysaccharide polymers secondarily modified with ethoxy containing groups. From a bioseparations perspective a major drawback of such single polymer, low salt, two-phase systems has been that proteins tend to partition without any selectivity into the upper (polymer poor) phase. Hence it has been written that “the water-EOPO system is therefore only suitable for partitioning of hydrophobic molecules (such as denatured proteins or tryptophan rich peptides or for solution concentration by selective water removal” (5). One possible academic solution was to modify EOPO polymers hydrophobically (6), however that may lead to detergency and other complications. It was recently discovered that EOPO polymers could be added directly to fermentation broths, taking advantage of the fermentation temperature and salts, to effect formation of a two-phase system where cell debris would be concentrated at the phase interface and soluble substances, such as proteins, would partition into the polymer-poor phase where they could then be readily filtered and chromatographed (4). This work was focussed on fermentations related to recombinant proteins in general and monoclonal antibody production in particular.
Protein Precipitation
While flocculation and precipitation (terms considered equivalent herein) have been proposed for cell debris removal (2) they also pose some attractiveness for processing bioprocess feeds pre-clarified using the classical approach of centrifugation followed by filtration (1, 2, 7-12). In those scenarios target purification may be effected by precipitation of contaminants such as host cell proteins (HCPs). Fortuitously most monoclonal antibodies, and many naturally occurring antibodies in blood, are net positively charged at pH 7, while many HCPs and nucleic acid contaminants are net negatively charged (7). Thus a polycationic polymer can be used to precipitate many HCPs and nucleic acid contaminants leaving net positively charged target monoclonal antibody (Mab) proteins in solution (8, 10). Alternatively one may wish to use net negatively charged polymers to precipitate net positively charged Mab targets and leave net negatively charged proteins and nucleic acids in solution (7-9). In both cases some form of filtration, centrifugation or other methodology is required to isolate the precipitate from the supernatant (11).
One advantage of precipitating an antibody or other target protein is that the target may be quite stable in the precipitate, and thus able to be stored intermittently during processing (12,13). A disadvantage is that dissociating the precipitate and re-solubilising the target protein (e.g. for follow-on purification via chromatography) can require diluting the protein which increases process volumes (e.g. 8). It may also require resuspension in buffers whose conductivity, pH or other properties are not commensurate with processing by chromatographic or other desired separation methods. An approach to purify biotechnical proteins such as Mabs and related antibody fragments (Fabs) using polycarboxylic acid in the presence of salts such as sodium citrate has recently been disclosed (7).
In the above examples pH was typically controlled to effect a situation where the substance to be precipitated had an opposite net charge to the precipitant it interacted with. Similar considerations apply if proteins such as antibodies are preferentially precipitated using salts such as ammonium sulphate, again using control of pH to ensure that antibodies are have opposite net charge to major contaminants (11). This helps ensure the protein is associated in complexes where it is charge neutral. When precipitants are uncharged, such as in the case of solvents like ethanol, or neutral (uncharged) polymers such as polyethylene glycol, control of solution pH to match the pH of isoelectric charge (pI) of the target protein is often very effective (12). This is because the precipitants typically bind water molecules and reduce target solvation.
Ethanol (14,15) and salts (22) have both been used to separate complex protein mixtures into several fractions by sequential precipitation, where one protein fraction after another is precipitated and recovered as the concentration of the precipitating agent is increased. This is possible because a) different proteins precipitate at different precipitant concentrations and b) once a particular protein has been precipitated, it is remains insoluble when the precipitant concentration is increased further. Precipitation with charged polymers as in (8,10) has however not been used for fractionation by sequential precipitation, as the precipitates tend to redissolve upon addition of an excess of precipitant polymer (8,9,10). This behaviour makes it essentially impossible to control a sequential precipitation process when two or more target proteins exhibit significantly different solubilities.
Plasma Protein Fractionation
Plasma is an invaluable source of relatively inexpensive therapeutic proteins; especially in developing countries. Every year several billion dollars of plasma proteins are isolated and sold for therapeutic uses. The major proteins of interest are those which are abundant in reasonably high concentration in plasma and include albumin, fibrinogen and various immunoglobulins such as those belonging to the IgG, IgA, and IgM classes. However other proteins such as clotting factors V, VII, VIII, IV and von Willebrand factor (vWF), as well as transferrin, fibronectin, and alpha-1-antitrypsin, are of growing commercial significance (14, 15).
TABLE 1Some Abundant Human Plasma ProteinsProteinMw (kDa)pIPlasma (g/l)Serum albumin695.635-55Immunoglobulin145-1906.5-9.514Fibrinogen3405.1-6.31.5-3  Transferrin805.6-6.02.3Factor VIII2805-60.20
In general donated blood is centrifuged to generate cell-rich fractions and plasma. A need for large scale centrifugation and sterility can limit the flexibility of such processes. Plasma is often stored frozen. Plasma proteins are purified based on chromatographic treatment of fractions which are first isolated using a general method termed Cohn Fractionation. It which was first developed by Cohn et al in the United States during World War II and was later modified by Kistler and Nitschmann and other scientists (14, 15). It is generally applied to large (e.g. 4000 L) volumes of thawing plasma to generate a cryoprecipitate which contains part of the fibrinogen as well as much of the Factor VIII and associated vWF. The supernatant is then subjected to a complex series of cold temperature precipitation steps in the presence of ethanol with various shifts of pH, temperature and conductivity (FIG. 1, redrawn from Ref 16). In modern plasma fractionation processes (e.g. FIGS. 2A and 2B, from Ref 15) these different precipitation steps yield fractions which are then subjected to further purification using chromatographic processes. In general the advantages of Cohn Fractionation include the fact that it can be applied to large volumes of plasma; that plasma protein fractions end up as precipitates which can be intermittently stored as needed; that some fibrinogen is removed early in the process so that it does not foul chromatography columns in follow-on separation steps; the ability of the processes to inactivate some bacteria, virus and perhaps prions; the ability of the process to be matched with solvent-detergent (SD) or related antiviral methods. Disadvantages include: a. The need to use large and fairly concentrated amounts of ethanol which presents both health and explosion/fire hazards leading to expensive processing equipment and facilities. b. The need for fine-tuned (+/−3° C.) temperature control which in the case of large liquid volumes further complicates processes and related equipment and facilities. c. Unit operations involving high concentrations of ethanol (up to 40%) and low pH as well as relatively long processing times which can denature some target proteins. d. Proteins such as fibrinogen and immunoglobulins have to be (partially) recovered as precipitates at different stages (e.g. FIGS. 2A and 2B). e. Protein recoveries can be quite poor e.g. 50% for immunoglobulins in some processes (above references). The alternative of using salt precipitation for fractionation (22) leads to very high salt contents in the recovered protein fractions, necessitating costly additional operations like dilution or diafiltration before further processing of these fractions. It may also lead to denaturation of the proteins. Fractionation using precipitation with non-ionic polyethylene glycol (PEG) and sodium chloride has also been attempted (23), but this requires very high concentrations of PEG and corrosive sodium chloride. In addition, this process is very pH-sensitive and careful adjustment of pH and conductivity is needed after each precipitant addition. It also needs complex additional processing to remove PEG from the proteins.
Given the above there is a need to identify simpler, robust and easily scaled alternatives to centrifuge-based blood cell isolation from plasma. There is also a need to identify simpler, easily scalable alternatives to Cohn Fractionation (14,15). It should also be noted that there is a need to identify rapid and simple methods to isolate plasma proteins for diagnostic and research purposes.